The steadily high mortality rates associated with melanoma are mainly due to the limitations of current therapies available for patients with advanced or metastatic disease. Immunotherapy is one of the few alternatives that provide more durable survival benefits for metastatic patients. Among the novel immunotherapeutic agents in metastatic melanoma treatment is ipilimumab, which stimulates T-cell mediated immune response by blocking CTLA-4, an antigen responsible for negative immunogenic signaling. Despite promising response compared to other immunotherapies, only ~ 30% of patients currently benefit from ipilimumab treatment. This indicates that there are other underlying host-related factors impacting the mechanism, under which ipilimumab blocks CTLA4-mediated immune inhibition. However, while molecular medicine would strongly benefit from such knowledge, no targetable molecular modulators of CTLA-4 blockade have been identified so far. In our study we hypothesize that the efficacy of ipilimumab/CTLA4 inhibition is modulated by yet unknown inherited (germline) genetic variation or somatic genetic alterations in activated T-cells affecting the pathways that regulate CTLA4 immune checkpoints. The overall goal of our study is to discover novel pharmacogenomic targets (germline and/or somatic) that modulate sensitivity/resistance to ipilimumab, eventually leading to modified drug designs in improved immunotherapies and other targeted treatments. To identify germline genetic variants that affect the molecular mechanisms of ipilimumab/CTLA-blockade, in this study we propose a pilot investigation utilizing deep sequencing of 396 genes involved in CTLA4 signaling pathway comparing two extreme clinical subsets: 50 ipilimumab-complete responders and 50 ipilimumab non-responders, followed by the validation stage in additional n>300 ipilimumab treated patients (Specific Aim 1). In parallel, the transcriptome sequencing of the same set of 396 genes will be performed to identify somatically acquired genetic alterations in CTLA4 signaling pathways in circulating CD4+/CD8+ T-cells (Specific Aim 2). The cost efficient design and novel analytical platform will allow for the integrative analysis, combining te germline and somatic data to identify genetic surrogates underlying the modulatory mechanisms of ipilimumab/CTLA4-blockade. We suggest that the novel actionable genetic alterations discovered in this study (somatic and germline), impacting efficacy of ipilimumab in stimulation of immune response, will not only offer utilization in improved drug designs and therapeutic decisions, but will likely point to novel molecular mechanisms triggering the application to other combined therapy approaches in treatment of metastatic melanoma. The pilot examination of genetic alterations in CTLA4-regulated immune checkpoint, as proposed, will also significantly widen our knowledge on other potentially critical molecular mechanisms that impact T-cell mediated immune response in general.